Gene Therapy: Difference Between DNA- and RNA-Directed Gene Therapy
The concept of gene therapy, or correcting the disease-causing faulty genes, was introduced several decades ago. The initial attempt was to use DNA-directed gene therapy where the DNA therapeutics and their carriers have to enter the nucleus to exert actions.
A newer form of gene therapy directed at RNAs, RNA interference (RNAi), was introduced in the last decade. RNAi therapeutics are short stretch double-stranded RNA that degrade the complementary mRNA, and thereby produce sequence-specific post-transcriptional gene silencing and correct the expression of faulty genes or production of disease-causing proteins. The RNAi process occurs in the cytoplasm. The two types of RNAi are small interfering RNA (siRNA) and microRNA (miRNA). Both use the same enzymes and proteins to produce gene silencing. A siRNA targets a single mRNA whereas miRNA may target 250-500 different mRNAs.
There are several major differences between DNA- and RNA-directed gene therapy. First, the formulations of DNA and RNAi vectors are different. For example, relatively large DNA lipoplexes (e.g., with a diameter ranging from 0.4 to 1.4 micron) show more efficient transfection in cultured cells compared to smaller lipoplexes. In contrast, RNAi vectors such as those disclosed in the instant disclosure are smaller in size, e.g., in the nanometer range. Second, for DNA-directed gene therapy, the DNA therapeutics must travel across the cell membrane and the cytosol to reach the nucleus, which involves the following multiple processes: (a) Binding of vectors to the cell. (b) Internalization of DNA therapeutics through endocytosis and endosome formation. (c) After internalization, the DNA-vector complexes (lipoplexes or polyplexes) are released from endosomes. (d) Cytoplasmic transport of endosomes may bring the complexes near the perinuclear region in such a way that the released DNA would have a greater chance to enter the nucleus. (e) Dissociation of lipoplexes, resulting in separation of DNA and vector. (f) Lipoplexes or polyplexes that are unable to escape from endosomes are likely to be degraded in lysosomes. (g) DNA released from lipoplexes or polyplexes into the cytoplasm may enter the nucleus via different, non-mutually exclusive, mechanisms. One, DNA may enter the nucleus during mitosis when the nuclear membrane breaks down. The second mechanism involves an active, energy-dependent nuclear transport into the nucleus. This transport requires the presence of specific sequences in the plasmid that mediate its interaction with transport proteins such as importins and other nuclear transport mediators. Three, lipoplex-filled endosomes may fuse directly with the nuclear membrane, permitting a direct entry of DNA into the nucleus. (h) After entering the nucleus, the DNA-therapeutic is degraded or integrated into the host chromatin.
In contrast, RNAi therapeutics exert their actions in the cytosol and do not require entering the nucleus or interaction with chromosomal DNA. However, the other barriers up to the step of being released as intact RNAi agent in the cytosol remain.
Due to the above differences, the efficiency of DNA- and RNA-directed gene therapy has different requirements. Increase of the transfection of DNA-therapeutics can occur at any one of the eight steps involved in a successful DNA gene therapy outlined above. In contrast, RNAi transfection can be increased by improving the delivery (including being released from the vector and endosomes) of RNAi to, and its expression in, the cytosol. These differences are substantive. For example, early release of DNA complex from endosomes is undesirable as the released DNA is subjected to degradation by cytoplasmic nucleases. In contrast, early release of RNAi from endosomes to the cytosol is desired as the released RNAi can then induce gene silencing. Similarly, pen-nuclear accumulation of DNA complex is desired for enhancing the DNA entry into the nucleus. In contrast, pen-nuclear accumulation is not necessary for RNAi. In other words, approaches and delivery systems that work in DNA-directed gene therapy may not be applicable to RNA-directed gene therapy, and vice versa.